Dab-4 linked nitroxide citrate, useful as mri contrast agent

ABSTRACT

A method of imaging a joint of a subject comprises administering to the subject an amount of a salt of DAB-4 linked nitroxide citrate effective to enhance an MRI image of the joint and taking an MRI image of said joint.

Osteoarthritis is the most common joint disease and is present in themajority of people who are over 60 years old (D. T. Felson. Epidemiologyof the rheumatic diseases, In, Arthritis and Allied Conditions: ATextbook of Rheumatology, 13th edition, (W. J. Koopman, ed.), Williamsand Wilkins, Baltimore, 1997). Osteoarthritis can involve almost anyjoint, but most commonly involves distal and proximal interphalangealjoints in the hands, hips, knees, and cervical and lumbosacral spine.Clinical features of osteoarthritis include pain in the involved jointswhich is aggravated by activity and relieved by rest, stiffness afterperiods of immobility, joint enlargement, and ultimately, functionalimpairment with joint instability and loss of motion.

Articular cartilage is a critical component of efficient, painless jointfunction. Osteoarthritis is characterized by progressive loss ofarticular cartilage. Recent advances in the understanding of thepathogenesis of osteoarthritis suggest that it is possible to developdrugs that will be able to reverse pre-existing abnormalities inarticular cartilage or prevent their progression, if therapy is startedearly during the course of the disease. This is the theory behind thedevelopment of Rofecoxide by Merck. As a result, there is a tremendousneed for a non-invasive technique that can accurately measure earlycartilage degradation and the progression of those abnormalities.

To date, radiography has been limited in its ability to accuratelyevaluate cartilage disorders, because it is limited to measuring thejoint space width—the distance between the bones (J. C. Buckland-Wright,D. G. Macfarlane, J. A. Lynch, M. K. Jasani, C. R. Bradshaw. Joint spacewidth measures cartilage thickness in osteoarthritis of the knee: highresolution plain films and double contrast macroradiographicinvestigation. Ann. Rheum. Dis. 54: 263-268, 1995); (J. C.Buckland-Wright, D. O. Macfarlane, S. A. Williams, R. J. Ward. Accuracyand precision of joint space width measurements in standard andmacroradiographs of osteoarthritic knees. Ann. Rheum. Dis. 54: 872-880,1995). Cartilage cannot be imaged directly by this method. The osseousabnormalities (such as osteophyte formation) that eventually developwith ostcoarthritis are not sensitive indicators of early disease, norare they reliable measures of disease progression (M. W. Spring, J. C.Buckland-Wright. Contrast medium imbibition in osteoarthritic cartilage.Br. J. Radiol. 63: 823-825, 1990). Over the past several years, a greatdeal of attention has focused on the use of advanced cross-sectionalimaging techniques, such as magnetic resonance (MR) imaging for theassessment of abnormalities in articular cartilage. Numerous MRsequences and techniques have been evaluated. Unfortunately, thesestudies have yielded variable, contradictory and often disappointingresults. As a consequence, there is no universally accepted “bestsequence or technique” for accurate MR imaging of articular cartilage.Inclusion of intraarticular injection of the contrast agent gadolinium(Gd)-DTPA has shown promise for the evaluation of proteoglycan statusand cartilage morphology. However, the images must be post-processedwith the calculation of T1 measurements, limiting the acceptance of thetechnique.

This invention describes a diagnostic method taking advantage of aparticular dendrimer-linked nitroxide, as a selective contrast agent toimprove the MR evaluation of cartilage status in humans; and/or todiagnose the presence of the above-described osteoarthritis and/orrelated diseases and conditions, e.g., those involving disorders of orloss of articular cartilage, e.g., in any joint, including thosementioned above. Specifically, a dendrimer-linked nltroxide with fourterminal nitroxide groups has been synthesized that exhibits highrelaxivity, thereby allowing MR contrast enhancement at clinicallyrelevant concentrations (e.g., those typically used for Gd-DTPA). Thisdendrimer-linked nitroxide, when injected into the joint directly,exhibits high in vivo stability, low toxicity and very high efficacy,diffusing into cartilage at a rapid rate, verifying its unexpectedlygood properties as an MR contrast agent to accurately evaluate cartilagestatus in humans and achieve the other effects described above andbelow.

Nitroxyl functionalized dendrimers are known. See, e.g., A. W. Bosmannet al., “Five Generations of Nitroxyl-Functionalized Dendrimers,”Macromolecules, American Chemical Society, Easton, US, vol. 30, No. 12,pp. 3606-3611, Jun. 16, 1997.

Bosmann's dendrimers are poly(propylene imine) dendrimers(DAB-dendr-(NH₂)_(n), where n=2, 4, 8, 16, 32 . . . ) functionalizedwith 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxy(3-carboxy-PROXYL)radicals. Herein, such compounds are abbreviated as DAB-4 linkednitroxide (wherein n in such dendrimer is 4,DAB-dendrimer-(NH-3-CO-PROXYL)₄), DAB-8 linked nitroxide (wherein n is8, DAB-dendrimer-(NH-3-CO-PROXYL)₈). This invention relates to the useof one set of these dendrimers, i.e., salts of DAB-4 linked nitroxide asMRI contrast agents for imaging joints.

Thus, it is an object of this invention to provide a method of MRIimaging of joints in a subject. It is also an object to provide an MRImethod of evaluating cartilage status in joints. It is further an objectto provide such methods achieving the characteristics outlined above andbelow.

The present invention provides such a method of imaging a joint byMR-imaging (“MRI imaging”) comprising imaging a joint of a subject(e.g., an animal, e.g., domestic animals (cats, dogs, etc.), humans,etc.) whose joint tissue contains a DAB-4 linked nitroxide citrate saltin an amount effective for MRI image enhancement. A preferred methodcomprises administering the salt of DAB-4 linked nitroxide to a subjectin need of MRI imaging of a joint and then MRI imaging such joint. Inanother aspect, the method comprises MRI imaging of a joint of a subjectto whom a DAB-4 linked nitroxide citrate salt has been administered. Inyet other aspects, such methods comprise evaluating cartilage status injoints, e.g., articular cartilage assessment.

The high usefulness of DAB-4 linked nitroxide citrate salt for enhancingMRI images of joints is unexpected in view of the prior art. Indiscussing the use of DAB-dendr-(NH₂)_(n) dendrimers functionalized with3-carboxy-PROXYL as MRI contrast agents for imaging joints, U.S. Pat.No. 6,991,778 requires that there be at least sixnitroxyl-functionalized 3-carboxy-PROXYL groups in such dendrimers(DAB-6), thereby excluding DAB-4 linked nitroxide salts of thisinvention from such use. However, surprisingly and unanticipated,despite their lower relaxivity than such dendrimers containing morenitroxyl-groups (e.g., Table 1 of '778), DAB-4 linked nitroxide citratesalt have overall characteristics overcoming this clear prior artprejudice, as well as that of Winalski et al., Osteoarthritis andCartilage (2008) 16, 815-822, and making them unexpectedly useful as MRIcontrast agents for joints, e.g., especially because of rapid diffusioninto joint tissue, yielding high quality images after only relativelyshort times after injection.

DAB-4 linked nitroxide and its salts can be prepared as presented in theexamples below or as described in Bosmann et al. above. Its salts, aswell, are conventionally preparable, e.g., analogously to the examplesbelow, e.g., by simply mixing the respective preferably organic acidwith DAB-4 linked nitroxide. Suitable salts other than the citrateswhich are operable are pharmaceutically acceptable salts, e.g., suchsalts prepared by conventional reaction with acids known for use in thepharmaceutical field. These include, but are not limited to, organicacids which may be selected from aliphatic, cycloaliphatic, aromatic,araliphatic, heterocyclic, carboxylic, and sulfonic classes of organicacids such as formic acid, acetic acid, propionic acid, glycolic acid,gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid,maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid,citric acid (for the citrates of the invention), aspartic acid, ascorbicacid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid,mandelic acid, embonic (pamoic) acid, phenylacetic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, salicyclic acid andthe like.

A particularly preferred salt is DAB-4 linked nitroxide citrate, e.g.,that formed with a 1:1 molar mixture of DAB-4 linked nitroxide andcitric acid. Other molar ratios of acid and DAB-4 linked nitroxide canalso be used, e.g., 2:1 to 1:2, etc., in dependence, e.g., on the numberof COOH groups or other acid moieties in the acid.

The term “joint” as used herein embraces any joint as the term embracesin the medical field, including joints of the fingers, toes, feet,hands, wrists, ankles, knees, elbows, neck, shoulders, back, hips, etc.Because the joints most frequently examined to evaluate their morphologyare the knee and shoulder joints, these are a preferred aspect of themethod of this invention.

The term “animal” or “subject” preferably means a human being. However,other animals, ordinarily mammals, and particularly valuable domesticmammals, such as dogs, cats, race horses, etc., are also embraced by theterm.

The term “pharmaceutically acceptable carrier” is entirely conventionaland includes all well-known carriers for conventional MRI contrastagents used to image joints, e.g., PBS conventionally sterilized forinjection, etc.

The dendrimer-linked nitroxide is ordinarily injected as a solution in anon-toxic injectable pharmacologically acceptable sterile aqueousvehicle, e.g., phosphate buffered saline (PBS) solution. The aqueousvehicle can also contain conventional amounts of other ingredientsconventionally present in solutions which are injected into a joint,e.g., local anaesthetic, anti-inflammatory agents, e.g., cortisone,antibiotics, stabilizing agents, suspending agents.

The concentration of the DAB-4 linked nitroxide citrate salt in aphosphate buffered saline solution is usually from about 1 to 10millimolar (mM), preferably from about 1 to 5 mM.

The amount of DAB-4 linked nitroxide citrate salt injected into thejoint, as is well known, is influenced by the volume of the joint void,the concentration of the DAB-4 linked nitroxide salt in solution (e.g.,phosphate buffered saline) injected into the joint. This is routinelydeterminable. Generally speaking, at a concentration of for example 5mM, a dose of about 15-25 mL injected into knee joint or shoulder jointcan be employed for DAB-4 linked nitroxide citrate salt, lower andhigher amounts not being excluded, according to conventionalconsiderations in humans. Knowing the in vitro image enhancing activityof DAB-4 linked nitroxide citrate salt per this invention and knowingthe dose required to obtain satisfactory MRI contrast enhancement in aparticular joint with, for example, Od-DTPA, a person knowledgeable inMRI can readily predict the optimum dose for obtaining the desireddegree of MRI contrast enhancement in a scan of that joint using DAB-4linked nitroxide citrate salt.

Times between the end of injection and beginning of the MR image areespecially advantageous for this invention. Typically, these are only30-60 minutes. These reflect a preferred aspect of this invention whereadvantage is taken of the short times required for diffusion of DAB-4linked nitroxide citrate salt into tissue sufficient for MRI imageenhancement thereof. Such advantageous time differentials are preferably30 minutes to 45 minutes. Of course, longer times are also within thescope of the invention.

Conventional MRI scanning techniques now employed extensively to obtainMR images of joints can be employed. See, e.g., DiChino, G., et al.,Radiology 1985 157:373-377; Portugal, F. H., High Technology August,1984, pp. 66-73: and Katzberg, R. W., The Contrast Media Manual”(Williams and Wilkins, Baltimore, Md., 1992). Buckland-Wright C. Whichradiographic techniques should we use for research and clinicalpractice? Best Pract Res Clin Rheumatol. February 2006; 20(1):39-55;131ark1.a.rid-Wright J C, Macfarlane D G, Williams S A, Ward R IAccuracy and precision of joint space width measurements in standard andmacrora.diographs of osteoarthritic. knees. Ann Rheum Dis. November1995; 54(11):872-880; Becht M P, Goodwin D W, Winalski C S, White L M.MRI of articular cartilage: revisiting current status and futuredirections. AJR Am J Roentgenol, October 2005; 185(4):899-914; Gold G E,Burstein D, Dardzinski B, Lang P, Boada F, Mosher T. MR.I. of articularcartilage in OA: novel pulse sequences and compositional/functionalmarkers, Osteoarthritis Cartilage. 2006; 14 Suppl A:A76-86.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments aretherefore to be construed as merely illustrative and not limitative ofthe remainder of the disclosure in any way whatsoever. Unless otherwiseindicated, all parts and percentages are by weight. The entiredisclosure of all applications, patents and publications cited hereinare incorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A: MRI T1W (weighted) image for DAB-8 linked nitroxide citrate wasobtained at 27 minutes after immersion of normal cartilage in thecontrast agent is shown.

FIG. 1B: MRI T1W (weighted) image for DAB-4 linked nitroxide citrate wasobtained at 29 minutes after immersion of normal cartilage in thecontrast agent is shown.

FIG. 1C: MRI T1 value map image for DAB-8 linked nitroxide citrate wasobtained at 27 minutes after immersion of normal cartilage in thecontrast agent is shown.

FIG. 1D: MRI T1 value map image for DAB-4 linked nitroxide citrate wasobtained at 29 minutes after immersion of normal cartilage in thecontrast agent is shown.

FIG. 1E: A pre-contrast T1 value map for normal cartilage is shown.

FIG. 2A: MRI Ti1 value map for DAB-4 linked nitroxide citrate incartilage, indicating high levels of contrast media and moreglycosaminoglycans (GAG) in the cartilage is shown.

FIG. 2B: Histology of the same specimen stain with Saframin-O/fast Greenis shown.

FIG. 2B: Depiction of high GAG content in the same tissue area s T1value map for DAB-4 linked nitroxide citrate is shown.

FIG. 3: Results for the MTT assay using the citrate, maleate andtartrate salts of DAB-4 linked nitroxide, DAB-8 linked nitroxide andMagnevist are shown.

FIG. 4: Results for the Picogreen assay using the citrate, maleate andtartrate salts of DAB-4 linked nitroxide, DAB-8 linked nitroxide andMagnevist are shown.

FIG. 5: Results for the MTT data normalized to □ gDNA/ml using thecitrate, maleate and tartrate salts of DAB-4 linked nitroxide, DAB-8linked nitroxide and Magnevist are shown.

FIG. 6: Data from the Modified Mankin scores for join degeneration usingDAB-4 linked nitroxide citrate, Magnevist and PBS, the latter two ascontrols is shown.

FIG. 7: Data from Synovial Inflammation scores for join degenerationusing DAB-4 linked nitroxide citrate, Magnevist and PBS, the latter twoas controls is shown.

FIG. 8: T1-weighted MRI image of a control knee joint is shown.

FIG. 9: T1-weighted MR image of a knee joint 10 minutes afterintraarticular injection of DAB-linked nitroxide citrate is shown.

EXAMPLE 1 Synthesis of DAB-4 Linked Nitroxide (Scheme 1)

In a solution of DMF (10 mL) containing DAB-4 (MW=316.5, 316 mg, 1mmoles) was added N,N-diisopropylethylamine (DIEA, MW=129, d=0.78, 5.0mmoles, 0.65 gm=0.83 mL) and3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (MW=186, 5.0 mmoles,0.93 gm) and the reaction was stirred at room temperature for ½ hr.Then, benzotriazole-1-yl-tris-(dimethylamino)phosphoniumhexafluorophosphate, (BOP, MW=442, 5.0 mmoles, 2.21 gm) was added. Thisreaction was stirred overnight, about 16 hr, at room temperature. Atthat point, a sodium chloride solution (50 mL), sodium bicarbonatesaturated (5 mL) and methylene chloride (100 mL) were added. The organiclayer was separated, dried over anhydrous sodium sulfate and evaporatedto dryness. The remaining residue was dried under high vacuum to removeremaining DMF. The resultant oil was chromatographed using silica gel(230-400 mesh, EMD Chemicals, distributed by VWR International,Bridgeport, N.J.). Elution with chloroform/acetone (40 mL/10 mL) broughtdown a small amount of a yellow compound, which was discarded. Changingto the following mixture: chloroform (25 mL)/methanol (25 mL) plustriethylamine (0.3 mL) afforded product one peak by TLC (silica gel,chloroform/methanol or alumina plates, chloroform/methanol). Mass spec(MW=989) confirms compound.

EXAMPLE 2 Synthesis of DAB-4 Linked Nitroxide (Scheme 1a)

In a solution of DMF (10 mL) containing DAB-4 (MW=316.5, 316 mg, 1mmoles) was added 3-carboxy-2,2S,5,5-tetramethyl-1-pyrrolidinyloxyl(MW=186, 5.0 mmoles, 0.93 gm) andO-(benzotrizol-yl)-N,N,N′N′-tetramethyluronim hexafluorophosphate (HBTU,MW=379, 1.9 gm, 5 mmoles). The reaction was stirred at room temperaturefor ½ hr. Then, N,N-diisopropylethylamine (DIEA, MW=129, d=0.78, 5.0mmoles, 0.65 gm=0.83 mL) was added. This reaction was stirred overnight,about 16 hr, at room temperature. At that point, a sodium chloridesolution (50 mL), sodium bicarbonate saturated (5 mL) and methylenechloride (100 mL) were added. The organic layer was separated, driedover anhydrous sodium sulfate and evaporated to dryness. The remainingresidue was dried under high vacuum to remove remaining DMF. Theresultant oil was chromatographed using silica gel (230-400 mesh, EMDChemicals, distributed by VWR International, Bridgeport, N.J.). Elutionwith chloroform/acetone (40 mL/10 mL) brought down a small amount of ayellow compound, which was discarded. Changing to the following mixture:chloroform (25 mL)/methanol (25 mL) plus triethylamine (0.3 mL) affordedproduct one peak by TLC (silica gel, chloroform/methanol or aluminaplates, chloroform/methanol). Mass spec (MW=989) confirms compound.

EXAMPLE 3 Synthesis of Citric Acid Salt of DAB-4 Linked Nitroxide(Schemes 1 and 1a)

Citric acid salt is prepared as follows. DAB-4-linked nltroxide (MW=989,0.74 gm, 0.7 mmoles) prepared in Example 1 or 2, dissolved in dry CHCl₃,and citric acid anhydrous (MW=192, 0.14 gm, 0.7 mmoles dissolved inabsolute ethanol are mixed together and evaporated to dryness. MW ofcitrate salt is 1,181.

EXAMPLE 4 Superiority of DAB-4 Linked Nitroxide Citrate for MRI Contrastof Cartilage

Comparison of DAB-4 linked nitroxide citrate and DAB-8 linked nitroxidecitrate for enhancement of normal cartilage. Cylindrical immature bovinecartilage plugs were imaged by MR at 7 Tesla initially in saline andthen in DAB-4 linked nitroxide citrate or DAB-8 linked nitroxidecitrate. T1-weighted (T1 W) images (TR=750 ms, TE=7 ms) and T1-valuemaps were performed. Images and T1 maps obtained ˜30 minutes afterimmersion in the contrast agents are shown in FIG. 1. Diffusion of thecontrast agent was restricted to occur only from the periphery and notfrom the top nor the bottom. The figures show:

a) DAB-8 linked nitroxide citrateT1W image 27 min after immersionb) DAB-4 linked nitroxide citrateT1W image 29 min after immersionc) DAB-8 linked nitroxide citrateT1 value map 27 min after immersiond) DAB-4 linked nistroxide citrateT1 value map 29 min after immersione) For comparison, a pre-contrast T1 value map for the cartilage

The DAB-8 linked nitroxide citrate shows much poorer penetration intothe cartilage. The enhancement is difficult to detect on the T1W images.The T1 value maps show the T1 values drop (contrast penetration) foronly a very thin rim of cartilage, only about ⅓ the distance for theDAB-4 linked nitroxide citrate.

EXAMPLE 5 Effectiveness of DAB-4 Linked Nitroxide Citrate to EnhanceContrast for Articular Cartilage Assessment as Measured by GAG Targeting

Cartilage-bone plug excised from a human tibial plateau that wasretrieved from total knee replacement surgery was equilibrated in DAB-4linked nitroxide citrate. T1 value map (as acquired for FIG. 1) iscompared to histology in FIG. 2:

a) T1 value map: orange color is lower T1 value indicating more contrastagent and more glycosaminoglycans (GAO) in the cartilage.b) Histology of the same specimen stained with Safranin-O/fast green.Comparison of FIG. 2a ) with FIG. 2b ) shows high GAG concentration inthe same areas indicated by DAB-4 linked nitroxide on the T1 value mapand the stain on the histology map:

EXAMPLE 6

Summary of Cytotoxicity Studies Comparing Salts of Dendrimer-LinkedNitroxides Based on DAB-4 and DAB-8 Dendrimer Cores with Magnevist andPositive and Negative Controls

Assays:

Modified MTT assay for metabolic activity (absorbance).

Quantitative picogreen for cell proliferation (gDNA/ml).

Assay Data Analysis:

Expressed as % of the positive controls for MTT and picogreen. MTTabsorbance measures then normalized to the ggDNA/ml generated by thepicogreen assay.

Cell Lures:

RCS-LTC chondrosarcoma cell line, Expresses hyaline-like chondrocytephenotype and reliably produces large, homogeneous cell populations

Compounds Tested:

Positive control—culture medium onlyNegative control—5 μM Staurosporine

Magnevist—1 mM, 5 mM, 7.5 mM, 10 mM

DAB-4 linked nitroxide Citrate—1 mM, 5 mM, 7.5 mM, 10 mMDAB-4 linked nitroxide Maleate—1 mM, 5 mM, 7.5 mM, 10 mMDAB-4 linked nitroxide Tartrate—1 mM, 5 mM, 7.5 mM, 10 mMDAB-8 linked nitroxide Citrate—1 mM, 5 mM, 7.5 mM, 10 mMDAB-8 linked nitroxide Maleate—1 mM, 5 mM, 7.5 mM, 10 mMDAB-8 linked nitroxide Tartrate—1 mM, 5 mM, 7.5 mM, 10 mM

Methods:

Cell cultures in 96 well plates. Each compound, at a fixedconcentration, was added in triplicate for each assay. MTT and picogreenassays in separate plates (assays interfere with each other). All platestreated together. Assays run at 48 hour timepoint. Microscopy performedto confirm findings.

Results for the MTT assay are shown in FIG. 3. At concentrations above 1mM, DAB-8 linked nitroxide shows less metabolic activity than positivecontrols (untreated), or DAB-4 linked nitroxide and Magnevist. DAB-4linked nitroxide showed less metabolic activity than positive controlsbut greater than for DAB-8 linked nitroxide. The citrate salt of DAB-4linked nitroxide showed more metabolic activity than the maleate ortartrate salts.

Results for the Picogreen assay are shown in FIG. 4. DAB-8 linkednitroxide citrate shows lower cell proliferation than positive control,(untreated), DAB-4 linked nitroxide maleate and Magnevist at allconcentrations. All DAB-4 linked nitroxide salts show a dose-dependenteffect. The 7.5 mM DAB-4 linked nitroxide tartrate appears to be anoutlier. Magnevist at 10 mM shows low cell proliferation. It is notclear if the lower cell proliferation is due to cell death or an initialdelay in proliferation.

Results for MTT data normalized to μgDNA/ml are shown in FIG. 5. This isa measure of the metabolic activity of the cells normalized to theconcentration of cells (μgDNA/ml), i.e. the “health” of the cells. DAB-4linked nitroxide salts perform similar to positive controls. DAB-8linked nitroxide salts show effects above 1 mM. Magnevist appearsabnormally high at 10 mM.

EXAMPLE 7 Toxicity Studies of DAB-4 Linked Nitroxide Citrate in RabbitJoints

The potential inflammatory and structural effects of PBS, Magnevist, andDAB-4 linked nitroxide citrate were assessed using a singleintra-articular injection model (data were taken from studies conductedby Arthroteq testing laboratory). Twelve female New Zealand Whiterabbits (2.7-3.3 kg) were randomly assigned to one of two study groupsthat received bilateral intra-articular injections with Magnevist (n=4)in the right stifle and sterile PBS in the left stifle, or DAB-4 linkednitroxide citrate (n=8, 0.3 mL of 10 mM) in the right stifle and sterilePBS in the left stifle (n=8, 0.3 mL).

Rabbits were euthanized two weeks post-injection. Both left and rightstifles were harvested and immediately fixed in 10% neutral bufferedformalin, then set for histological processing. Five-micron thick slidesfrom each knee were stained with Hematoxylin & Eosin and Safranin-O.

No animals presented clinical signs of inflammation or lameness duringthe study. Modified Mankin scoring did not uncover any signs ofstructural damage and/or degenerative response due to Magnevist, DAB-4linked nitroxide citrate, or PBS injections (FIG. 6). Custom synovialinflammation scoring reflected the absent of an inflammatory response inall stifles and no difference between Magnevist or DAB-4 linkednitroxide and the PBS-injected stifles were observed (FIG. 7).

FIG. 6: No differences were detected in the Modified Mankin subscores ortotal score between any treatment groups as assessed by ANOVA. All testswere performed at a significance level of p<0.05. Mean cartilagedegenerative changes assessed by Modified Mankin Scoring (Mean±S.D.) forall study groups. A higher score (maximum of 19) indicates greater jointdegeneration. Mean subscores for a naïve group of age-matched rabbitsnormally fall in a range between 0 and 1.0. Total Mankin Score for anaïve group of age-matched rabbits normally falls in a range between 0and 3.0.

FIG. 7: No differences were detected in the Synovial Inflammationsubscores or total score between any treatment groups as assessed byANOVA. All tests were performed at a significance level of p<0.05.Average scores (Mean±S.D.) for Synovial Inflammation for all studygroups. A higher score indicates greater inflammation. Only two of theseven sub-categories presented non-zero scores. A mean score between 0and 1.0 is typical for naïve age-matched rabbits.

EXAMPLE 8 MRI Images of a Knee Joint Using DAB4 Linked Nitroxide Citrate

Coronal T1-weighted MR images of a human knee were taken in a humancadaver. The cartilage surfaces are poorly differentiated as shown inFIG. 8. Coronal T1-weighted MR contrast images were also taken ex vivoin a human cadaver 10 minutes after intraarticular injection of 25 ml ofa 5 mM solution of DAB-4 citrate diluted in PBS. As shown in FIG. 9, thejoint fluid (asterisk) is now bright and outlines the cartilagesurfaces. A partial thickness cartilage defect (arrow) is easilyidentified. All images were performed on a Siemens Skyra 3 Tesla magnetusing a 15 channel transmit/receive QED knee coil. (14 cm FOV (field ofview); 3 mm thick slice; 384×384 matrix; TR 800 ms; TE 8.6 ms; Turbofactor of 2; receiver bandwidth 350 Hz/pixel.)

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1-12. (canceled)
 13. A compound comprising: DAB-4 linked nitroxidecitrate.
 14. The compound of claim 13 wherein the citrate salt is a 1:1molar ratio citric acid:DAB-4 linked nitroxide.
 15. A pharmaceuticalcomposition comprising the compound of claim 13 and a pharmaceuticallyacceptable carrier.
 16. The pharmaceutical composition comprising theDAB-4 linked nitroxide citrate of claim 14 and a pharmaceuticallyacceptable carrier.